The current study was conducted to investigate the effects of in ovo injection of recombinant clostridium NetB toxin plus Eimeria profilin proteins in combination with Montanide adjuvants in modulating immune system in chickens infected for experimental necrotic enteritis (NE) disease. First, the safety of adjuvants was tested for in ovo injection in broiler eggs at 18 days of incubation. In a second step, broiler eggs were injected with 100μl of PBS, profilin, profilin plus NetB, profilin plus NetB/Montanide IMS OVO adjuvant (OVO). Intestinal lesion scores, body weight gains, Net-B toxin antibody levels, and proinflammatory cytokine and chemokine levels were measured as outcomes of protection following oral co-infection with C. perfringens and Eimeria maxima. Birds in ovo vaccinated with recombinant profilin plus NetB proteins/OVO showed significantly increased body weight gains and reduced gut lesions compared with the profilin-only group, respectively. Greater NetB toxin antibody titers were observed in the profilin plus NetB/OVO group compared with the other three vaccine groups. Finally, decreased levels of gene transcripts encoding interleukin-8, tumor necrosis factor superfamily 15, and TNF-α factor were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with OVO compared with profilin protein alone bird. These results suggest that in ovo injection with recombinant profilin plus NetB proteins in combination with Montanide IMS adjuvants enhances protective immunity against experimental NE disease.